Coupled cofactor-dependent enzymatic reaction systems in aqueous media

ABSTRACT

The present application relates to a reaction system in which chemically valuable compounds can be obtained in high enantiomer concentrations with the aid of a coupled enzymatically operating transformation process. The coupled enzymatic reaction system comprises a cofactor-dependent enzymatic transformation of an organic compound and an enzymatic regeneration of the cofactor, wherein the reaction system operates in aqueous solution with an amount of substrate above the solubility limit thereof. In the preferred embodiments, an alcohol dehydrogenase is the cofactor-dependent enzyme, and the regeneration of the cofactor (e.g. NADH or NADPH) is acheved by means of formate dehydrogenase.

The present invention relates to a coupled enzymatically operatingreaction system for reduction of carbonyl compounds, which isdistinguished in that it is carried out in an emulsion. In particular,the invention relates to a reaction system comprising acofactor-dependent enzymatic transformation of an organic compound,preferably the reduction of a carbonyl compound, wherein the cofactor isregenerated enzymatically in the same system.

The production of optically active organic compounds, e.g. alcohols andamino acids, by a biocatalytic route is increasingly gaining importance.The coupled use of two dehydrogenases with cofactor regeneration hasemerged as a route for the large-scale industrial synthesis of thesecompounds (DE19753350).

In situ regeneration of NADH with the NAD-dependent formatedehydrogenase in the reductive amination of trimethylpyruvate to giveL-tert-leucine (Bommarius et al. Tetrahedron Asymmetry 1995, 6,2851-2888).

In addition to their catalytic property and efficiency, the biocatalystsefficiently employed in an aqueous medium furthermore have the advantagethat in contrast to a large number of synthetic metal-containingcatalysts, the use of metal-containing starting substances, inparticular those which contain heavy metals and are therefore toxic, canbe dispensed with. The use of expensive and furthermore hazardousreducing agents, such as, for example, borane, in the case of asymmetricreduction can also be dispensed with.

Nevertheless, difficulties occur in the reaction of substrates which arepoorly water-soluble. This affects in particular the preparation ofalcohols from hydrophobic carbonyl compounds, in which the substratesolubility is often below 10 mM. Similar difficulties exist in the caseof poorly water-soluble products. A solution which is conceivable inprinciple would be to carry out the biocatalytic reduction in a polarorganic solvent or a resulting homogeneous aqueous solution thereof. Inthis case, both the enzymes and the substrate and, where appropriate,the product should be water-soluble. A general disadvantage of a directpresence of an organic solvent, however, is the considerable reductionwhich generally occurs in the enzyme activity under these conditions(see e.g. Anderson et al., Biotechnol. Bioeng. 1998, 57, 79-86). Inparticular, FDH as the only formate dehydrogenase employed hitherto onan industrial scale and accessible in commercial amounts unfortunatelyhas a high sensitivity towards organic solvents. This also manifestsitself in the comparison examples 1 using DMSO, sulfolane, MTBE,acetone, isopropanol and ethanol as the organic solvent component inadded amounts of in each case 10% (see FIG. 1).

Various set-ups are known to solve this problem relating tostabilization of the formate dehydrogenase from Candida boidinii in thepresence of organic solvents, e.g. carrying out reactions by theadditional use of surfactants as surface-active substances.Disadvantages here, however, are the rate of reaction, which is reducedby about a factor of 40 (!), and the inhibition of formate dehydrogenasewhich occurs (B. Orlich et al., Biotechnol. Bioeng. 1999, 65, 357-362).The authors furthermore note that because of the low stability of thealcohol dehydrogenase employed, a reduction process under theseconditions of a microemulsion is not economical. In addition, there is afurther problem in the working up, in which the resulting product mustbe separated from the surfactant, which has often proved to be not atrivial matter.

A possibility in principle also consists of carrying out enzymaticreactions or oxidations in a two-phase system. Here however—analogouslyto the abovementioned destabilizing effects in the presence of organicwater-soluble solvents—only a particular class of organic solvents,namely those with a very hydrophobic character, such as, for example,heptane and hexane, has proved to be suitable. On the other hand,stability studies with other nonpolar solvents, such as toluene, butabove all with typical solvents such as MTBE and ethyl acetate, showed adrastic decrease in the activity of the formate dehydrogenase fromCandida boidinii even in a very short service life (H. Groger et al.,Org. Lett. 2003, 5, 173-176). In the presence of heptane and hexane, incontrast, the reaction can indeed be carried out, but the solubility ofthe ketone substrates in these solvents is often limited.

A further possibility in principle for carrying out biocatalyticreactions consists of the use of immobilized enzymes in the organicsolvent or the use of enzymes in a homogeneous solution comprising waterand a water-miscible organic solvent. However, these techniques in whichdirect contact occurs between the organic solvent and enzyme are limitedto a few enzyme classes, in particular hydrolases. It is thus noted inDE4436149 that the “direct presence of organic solvents (water-miscibleor water-immiscible) is tolerated by only a few enzymes which belong tothe class of hydrolases”. A few further examples from other enzymeclasses have indeed since become known (thus, inter alia,oxynitrilases), but the statement made in DE4436149 is still applicableto the majority of enzymes. An efficient immobilization of the FDH fromCandida boidinii is thus not known. Rather, for example, it is knownwith the Eupergit method, as a standard tool of industrialimmobilization, that the residual activity of this FDH afterimmobilization is <20%, which is too low for an industrial utilization.Furthermore, the immobilization itself is associated with additionalcosts due to the immobilization step and the immobilization materials.

Industrially, processes have therefore been developed which avoid thepresence of organic solvents because of the risk of deactivation ordenaturing of the enzymes. DE4436149 thus describes a process in whichthe product is extracted from the reaction solution into an organicsolvent through a membrane, in particular a hydrophobic membrane, whichis permeable to the product. Compared with a standard process in astirred tank reactor, however, this process requires significantly moretechnical outlay, especially since the organic membranes required arealso an additional cost factor. Furthermore, this method is suitableonly for continuous processes. In addition, the disadvantage inprinciple of carrying out the reaction at low substrate concentrationsalso cannot be avoided with this method. Accordingly, the substrateconcentrations are below the solubility limit, which for most ketones is10 mM or considerably lower. However, substrate concentrations of 100 mMor above would be desirable for an industrial reaction.

Summarizing, it can be said that thus no process which helps to bypassthe abovementioned disadvantages is known.

The object of the present invention was therefore to provide apossibility such that, in particular, poorly water-soluble organiccompounds can be rendered accessible to a coupled cofactor-dependentenzymatic reaction to an adequate extent such that the possibility canbe used on an industrial scale under, in particular, economically andecologically advantageous conditions.

This object is achieved according to the claims. Claims 1 to 10 relateto a reaction system which operates according to the invention. Claims11 and 12 protect a process according to the invention and claims 13 and14 protect the advantageous use of the reaction system.

By providing a coupled enzymatic reaction system comprising acofactor-dependent enzymatic transformation of an organic compound andan enzymatic regeneration of the cofactor in a purely aqueous solventsystem without addition of surfactant, wherein the substrate is employedin the enzymatic transformation in an amount of at least 50 mM per litreof water, as long as this does not fall below the solubility limit ofthe substrate, the stated object is achieved in particular in asurprising, in no way foreseeable and, according to the invention,particularly advantageous manner. In contrast to the opinion which canbe deduced from the prior art, in particular in view of the feareddramatic decreases in the activity of the enzymes and here in particularin that of the formate dehydrogenase from Candida boidinii in thepresence of organic components with a log P value of <3.5 (under whichalso most of the substrates and products fall), it is possible,surprisingly and in spite of the direct presence of such organiccomponents (substrates/products), to operate the coupled enzymaticreaction system without a significant loss in activity (of one) of theenzymes. Comparison example 2 underlines this surprising effect;according to this drastic decrease in activity observed in comparisonexample 2, with a virtually complete loss in activity of the FDH withinonly a few hours, it would have been expected that no significantconversions result under the reaction conditions according to theinvention.

It is thus advantageous that an emulsion or a suspension is present inthe reaction system at least initially. The amount of substrate employedis particularly preferably 50 to 1,500 mM, very particularly preferably100 to 1,000 mM, and extremely preferably 100 to 500 mM per litre ofwater, as long as this does not fall below the solubility limit of thesubstrates.

The cofactor-dependent transformation is advantageously the reaction ofan oxidoreductase. Carbonyl compounds, in particular aldehydes orunsymmetric ketones, can advantageously serve as the substrate for thistype of conversion. These are reduced in an advantageous manner toenantiomerically enriched alcohols.

However, it is also possible to employ an alcohol compound as thesubstrate, in particular a primary or a chiral secondary alcohol, whichis then oxidized accord ingly. The nature of the reactions is diverseand includes all types of redox reactions. The present reaction systemis particularly suitable for the reduction of carbonyl compounds to formenantiomerically enriched alcohols. In this context, both the reductionof aldehydes to form primary alcohols (for this see also example 7) andthe asymmetric reduction of unsymmetric ketones (for this see examples 3to 6) are of particular importance.

The reaction system can be operated with any cofactor-dependentoxidoreductase, where the cofactor is consumed by the oxidoreductase andcan be regenerated by a second enzymatic system, that is to say thesystem is a coupled enzymatic system. Further suitable enzymes of thistype can be found in the literature (Enzyme Catalysis in OrganicSynthesis; Ed.: K. Drauz, H. Waldmann, Vol. I and II, VCH, 1995).

An alcohol dehydrogenase or amino acid dehydrogenase has proved to be anenzyme which it is preferable to employ.

The nature of the regeneration of the cofactor primarily depends on thecofactor employed itself. Various methods of cofactor regeneration canbe found in the abovementioned literature. Under the given frameworkconditions of solvent, enzymes and space/time yield, the expert has afree choice of the regeneration medium. In general, in respect of NAD+asthe cofactor (in oxidation reactions) an NADH oxidase from e.g.Lactobacillus brevis or L. kefir is suitable (DE10140088). In the caseof reduction reactions, regeneration of the cofactor NADH by a formatedehydrogenase has furthermore also proved to be very successful. The useof the formate dehydrogenase from Candida boidinii is particularlyadvantageous in this connection.

The cofactors which are the most usual and operate most economicallyunder the reaction conditions are preferably used as cofactors. Theseare, in particular, cofactor NADH or NADPH.

The present application also provides a process for the enzymatictransformation of an organic compound using the reaction systemaccording to the invention. The process is preferably the preparation ofan enantiomerically enriched organic compound, preferably a primary or achiral secondary alcohol.

The process procedure can be implemented as desired by the expert, withthe aid of the reaction system described and the examples described inthe following. The conditions which are otherwise known for theenzymatic reaction are set accordingly under the given frameworkconditions.

The reaction can thus preferably be carried out at temperatures of 10 to80° C., preferably 20 to 60° C., and very preferably 20 to 40° C. Whensetting the temperature, the expert will be guided by frameworkconditions such as e.g. speed of the reaction, yield, enzyme stabilityand by-product spectrum.

When the reaction is complete, the now homogeneous or heterogeneousreaction mixture can advantageously be treated in a manner in which thereaction mixture is separated into an aqueous and an organic phase, ifappropriate by addition of an organic solvent, and the desired productis isolated from the organic phase.

The invention also relates to a device for the transformation of organiccompounds comprising a reaction system according to the invention.

Devices which are advantageously to be employed are, for example, astirred tank or cascades of stirred tanks.

One aspect of the invention is also the use of the reaction systemaccording to the invention for the enzymatic transformation of organiccompounds or for diagnosis or analysis. In this context, the enzymatictransformation of an organic compound is preferably carried out with theformation of enantiomerically enriched products.

According to the invention, coupled enzymatic system is understood asmeaning that an enzymatic transformation of an organic compound proceedswith the consumption of a cofactor and the cofactor is regenerated insitu by a second enzymatic system. As a result, this leads to areduction in the use of expensive cofactors, since these have to beemployed only in catalytic amounts—based on the total conversion.

It is particularly surprising here that in spite of current doctrine thetwo enzymes employed are not impaired by the presence of the emulsionand it is thus possible to prepare the desired products in very goodspace/time yields.

As has been shown, for both aldehydes and ketones—in contrast to mostorganic solvents (see comparison examples), which lead to rapiddeactivation of the FDH employed—outstanding stability properties of theenzymes, in particular the very unstable formate dehydrogenase, can alsostill be observed after several days even at high substrateconcentrations. In addition, the rapid course of the reaction, whichtakes place at a rate similar to that at very low ketone concentrationsin purely aqueous solution (that is to say under theoretically the mostoptimum conditions), is very surprising. This rapid formation rate underthe process conditions was in no way at all to be expected, last but notleast also in view of the considerable decreases in activity on additionof ketone substrates in small amounts of <15 mM (see comparison example2). Rather, on the basis of these considerable losses in activity evenin the presence of small amounts of ketone it would have been expectedthat if the substrate concentration is increased further, no or only alow conversion takes place. In contrast to this expectation, the desiredreaction surprisingly not only proceeds extremely rapidly under theprocess conditions, but also surprisingly leads to a completeconversion.

The results with the new reaction system according to the invention arereproduced in the experimental part. The comparison examples with otherorganic solvents are shown in FIG. 1.

The process is carried out both with the wild-type of the formatedehydrogenase from Candida boidinii and with a form of this enzymemodified by genetic engineering (DE19753350). As stated, NADH ispreferably employed as the cofactor. For the experimental studies, forexample, an ADH from Rhodococcus, preferably Rhodococcus erythropolis,can be employed as the ADH component.

In general, the enzymes employed can be used for the reaction in a cellfree native or recombinantly prepared form purified as desired. In thiscontext, crude extracts are also preferably employed.

A main advantage of this process is the simplicity of the process. Thus,it comprises no expensive process steps, and the process can be carriedout in the preferred batch reactors. Likewise, in contrast to earlierprocesses no special membranes which separate the aqueous medium fromthe organic medium are required. The surfactant additions required insome processes to date are also omitted in this process. This was not tobe seen from the prior art and nevertheless makes the present processextremely advantageous.

Moreover, the further downstream processing is extremely simple. Asimple extraction with a water-insoluble organic solvent leads to asimple method of isolation of the product formed. The possiblequantitative conversion moreover renders possible the existence of acrude product which is already highly pure—after evaporation of theorganic extraction agent in vacuo. An expensive purification of theproduct from a (possibly also) high-boiling substrate is accordinglydispensed with.

Enantiomerically enriched or enantiomer-enriched describes the fact thatone optical antipode is present in a mixture with its other to >50%.

The structures shown relate to all the possible diastereomers and, inrespect of a diastereomer, to the two possible enantiomers of thecompound in question which fall under this.

The process according to the invention is illustrated by the examplesdescribed below.

EXPERIMENTAL PART Example 1 Comparison Examples of FDH Activities

2.72 g (0.8 mol/l) sodium formate and 1.14 g (0.1 mol/l) di-potassiumhydrogen phosphate trihydrate are weighed out and are dissolved in 40 mlof completely demineralized H₂O. The pH of the solution is adjusted to8.2 with ammonia solution (25%) and formic acid (100%) or appropriatedilutions. The solution is then transferred to a 50 ml volumetric flaskand topped up with completely demineralized H₂O. Separately to this,71.7 mg (4 mmol/l) NAD⁺ trihydrate are weighed out and dissolved inapprox. 20 ml of completely demineralized H₂O. The pH of the solution isadjusted to 8.2 with ammonia solution (25%) and formic acid (100%) orappropriate dilutions. The solution is then transferred to a 25 mlvolumetric flask and topped up with completely demineralized H₂O. Ineach case 500 μl of the substrate solution and of the NADH solution arethen mixed in the 1 cm cell used for the measurement. After addition of10 μl of the enzyme solution, a 10% solution of an organic solvent (seetable) in water being employed as the solvent, the mixture is shakenbriefly, the cell is placed in the photometer and recording of the datais started. The enzyme solution is added only directly before the startof the measurement. The activities of the enzymes are determined aftercertain intervals of time by photometric detection of the reaction ofNAD⁺ to give NADH. The photometric measurement was carried out at atemperature of 30° C. and a wavelength of 340 nm with a measurement timeof 15 min. The results are shown in the following in table 1 and table2. TABLE 1 Enzyme activity of the FDH in U/ml as a function of thesolvent and time Butanol MEK DMSO THF Sulfolane Acetonitrile TimeActivity Activity Activity Activity Activity Activity [d] [U/ml] [U/ml][U/ml] [U/ml] [U/ml] [U/ml] 0.000 0.5262 0.0058 0.7965 0.8492 0.00280.7961 0.042 0.0006 0.0011 0.7880 0.4357 0.0003 0.4494 0.125 0.77940.0414 0.0840 1.097 0.2669 0.0008 2.035 0.2331 2.896 0.2201 5.927 0.17637.885 0.1404 9.948 0.1205 13.073 0.0915 14.892 0.0717 16.875 0.054019.938 0.0355

TABLE 2 Enzyme activity of the FDH in U/ml as a function of the solventand time Acetone Ethanol Time Activity Activity [d] [U/ml] [U/ml] 0.0000.8355 0.8491 0.042 0.7402 0.7689 0.750 0.5893 0.6367 1.000 0.54260.5933 1.875 0.3484 0.4687 2.760 0.2691 0.3510 3.781 0.2004 0.2814 4.6460.1614 0.2240 5.875 0.1325 0.1736 6.778 0.0987 0.1486 7.792 0.07940.1277 8.729 0.0610 0.0998 11.750 0.0333 0.0536 13.726 0.0421

Example 2 Comparison Example; Measurement of the FDH Long-TermActivities in the Presence of 2′,3-dichloroacetophenone as an Additive

The activities of the formate dehydrogenase were measured in accordancewith the procedure described in comparison example 1, but without theuse of an organic solvent. In this context, various amounts of ketoneconcentration of 2′,3-dichloroacetophenone were added as an additive.The resulting course of the stability is shown in FIG. 2. When2′,3-dichloroacetophenone was used, a rapid deactivation took placewithin 5 hours at substrate concentrations of >10 mM.

Example 3 Reaction with 2-chloroacetophenone at 250 mM

A reaction mixture, comprising ortho-chloroacetophenone(2-chloroacetophenone; 250 mM), as well as NADH (0.04 equivalent, basedon the ketone), and sodium formate (5.5 equivalents, based on theketone) at enzyme amounts of 60 U/mmol of an (S)-ADH from R.erythropolis (expr. in E. coli) and 60 U/mmol of a formate dehydrogenasefrom Candida boidinii (double mutants: C23S, C262A; expr. in E. coli),is stirred at a reaction temperature of 30° C. over a period of 72 hoursin 50 ml of a phosphate buffer (100 mM; pH 7.0). Samples are takenduring this period of time and the particular conversion is determinedvia HPLC. After 72 hours, complete conversion of the ketone to thedesired alcohol was found. The organic components are then extractedwith 2×50 ml methyl tert-butyl ether, the aqueous phase is discarded andthe organic phase is dried. The filtrate which results after filtrationis freed from the readily volatile constituents in vacuo and theresulting residue is investigated in respect of the formation rate byanalysis via HPLC and ¹H nuclear magnetic resonance spectroscopy. Aformation rate of >99% was determined (FIG. 3).

Example 4 Reaction with 2-chloroacetophenone at 400 mM

A reaction mixture, comprising ortho-chloroacetophenone(2-chloroacetophenone; 400 mM, based on the total volume), as well asNADH (0.04 equivalent, based on the ketone), and sodium formate (5.5equivalents, based on the ketone) at enzyme amounts of 60 U/mmol of an(S)-ADH from R. erythropolis (expr. in E. coli) and 60 U/mmol of aformate dehydrogenase from Candida boidinii (double mutants: C23S,C262A; expr. in E. coli), is stirred at a reaction temperature of 30° C.over a period of 46.5 hours in 12 ml of a phosphate buffer (100 mM; pH7.0), the total volume being 20 ml. Samples are taken during this periodof time and the particular conversion is determined via HPLC. After 46.5hours, complete conversion of the ketone to the desired alcohol wasfound via HPLC (FIG. 4).

Example 5 Reaction with 4-chloroacetophenone at 250 mM

A reaction mixture, comprising para-chloroacetophenone(4-chloroacetophenone; 250 mM, based on the total volume), as well asNADH (0.04 equivalent, based on the ketone), and sodium formate (5.5equivalents, based on the ketone) at enzyme amounts of 60 U/mmol of an(S)-ADH from R. erythropolis (expr. in E. coli) and 60 U/mmol of aformate dehydrogenase from Candida boidinii (double mutants: C23S,C262A; expr. in E. coli), is stirred at a reaction temperature of 30° C.over a period of 46.5 hours in 15 ml of a phosphate buffer (100 mM; pH7.0), the total volume being 20 ml. Samples are taken during this periodof time and the particular conversion is determined via HPLC.

After 46.5 hours, a conversion of >99% of the ketone to the desiredalcohol was found (FIG. 5).

Example 6 Reaction with 2′,3-dichloroacetophenone at 300 mM

A reaction mixture, comprising alpha,meta-dichloroacetophenone (2′,3-dichloroacetophenone; 300 mM, based on the total volume), as well asNADH (0.04 equivalent, based on the ketone), and sodium formate (5.5equivalents, based on the ketone) at enzyme amounts of 60 U/mmol of an(S)-ADH from R. erythropolis (expr. in E. coli) and 60 U/mmol of aformate dehydrogenase from Candida boidinii (double mutants: C23S,C262A; expr. in E. coli), is stirred at a reaction temperature of 30° C.over a period of 46.5 hours in 14 ml of a phosphate buffer (100 mM; pH7.0), the total volume being 20 ml. Samples are taken during this periodof time and the particular conversion is determined via HPLC. After 46.5hours, a conversion of >98% of the ketone to the desired alcohol wasfound (FIG. 6).

Example 7 Reaction with Cinnamaldehyde at 100 mM

A reaction mixture, comprising cinnamaldehyde (100 mM, based on theamount of buffer employed), as well as NADH (0.2 equivalent, based onthe ketone), and sodium formate (5.0 equivalents, based on the ketone)at enzyme amounts of 20 U/mmol of an (S)-ADH from R. erythropolis (expr.in E. coli) and 20 U/mmol of a formate dehydrogenase from Candidaboidinii (double mutants: C23S, C262A; expr. in E. coli), is stirred ata reaction temperature of 30° C. over a period of 24.25 hours in 10 mlof a phosphate buffer (100 mM; pH 7.0). Samples are taken during thisperiod of time and the particular conversion is determined via HPLC.

After 24.25 hours, a conversion of >95% of the aldehyde to the desiredalcohol was found (FIG. 7).

1-14. (canceled)
 15. A coupled enzymatic reaction system, comprising: a) a cofactor-dependent enzymatic transformation of an organic compound; and b) an enzymatic regeneration of the cofactor; wherein: i) said cofactor-dependent enzymatic transformation of step a) and said enzymatic regeneration of step b) are carried out in a purely aqueous solvent system in the absence of surfactant; and ii) the initial concentration of substrate used for the enzymatic transformation is at least 50 mM and wherein the concentration of said substrate is higher than or equal to its solubility limit.
 16. The reaction system of claim 15, wherein an emulsion or a suspension is initially present in the reaction system.
 17. The reaction system of claim 15, wherein the initial concentration of said substrate is between 100 and 1,000 mM, and wherein the concentration of said substrate is higher than or equal to its solubility limit.
 18. The reaction system of claim 15, wherein said substrate is a carbonyl compound or an alcohol.
 19. The reaction system of claim 18, wherein said substrate is an aldehyde, unsymmetric ketone, primary alcohol, or chiral secondary alcohol.
 20. The reaction system of claim 15, wherein NADH or NADPH is used as a cofactor.
 21. The reaction system of claim 15, wherein reactions are carried out at a temperature of between 10 and 80° C.
 22. The reaction system of claim 15, wherein a dehydrogenase is employed as the enzyme for the cofactor-dependent enzymatic transformation of step a).
 23. The reaction system of claim 22, wherein said dehydrogenase is an alcohol dehydrogenase.
 24. The reaction system of claim 15, wherein regeneration of cofactor takes place by means of a formate dehydrogenase.
 25. A process for producing organic compounds comprising the reaction system of claim
 15. 26. The process of claim 15, wherein the reaction mixture is separated into an aqueous and an organic phase by the addition of an organic solvent, and the desired product is isolated from the organic phase.
 27. The reaction system of claim 16, wherein said initial substrate concentration is between 100 and 500 mM, and wherein the concentration of said substrate is higher than or equal to its solubility limit
 28. The reaction system of claim 27, wherein a carbonyl compound or an alcohol is employed as the substrate.
 29. The reaction system of claim 28, wherein said substrate is an aldehyde, an unsymmetric ketone, a primary alcohol, or a chiral secondary alcohol.
 30. The reaction system of claim 28, wherein NADH or NADPH is used as a cofactor.
 31. The reaction system of claim 30, wherein reactions are carried out at a temperature of between 20 and 40° C.
 32. The reaction system of claim 31, wherein a dehydrogenase is employed as the enzyme for the cofactor-dependent enzymatic transformation of step a).
 33. The reaction system of claim 32, wherein said dehydrogenase is an alcohol dehydrogenase.
 34. The reaction system of claim 33, wherein regeneration of cofactor takes place by means of formate dehydrogenase.
 35. A process for producing organic compounds comprising the reaction system of claim
 34. 36. The process of claim 35, wherein the reaction mixture is separated into an aqueous and an organic phase by the addition of an organic solvent to the reaction mixture. 